Stroke is a debilitating disease which affects more than 400,000 persons per year in the United States and is the third most common cause of death in the United States. In addition one-half of neurology inpatients have stroke related problems. At current trends, this number is projected to jump to one million per year by the year 2050. When the direct costs (care and treatment) and the indirect costs (lost productivity) of strokes are considered together, strokes put a burden of $43.3 billion per year on the society of the United States alone. About ⅓ of patients die in the first three months, ⅓ remain with severe disabilities, and only ⅓ recover with acceptable outcome. In 1990 cerebrovascular diseases were the second leading cause of death worldwide, killing over 43 million people world wide. Thus, from a public health perspective, stroke is one of the most relevant diseases.
Stroke is characterized by the sudden loss of circulation to an area of the brain, resulting in a corresponding loss of neurologic function. Also called cerebrovascular accident or stroke syndrome, stroke is a nonspecific term encompassing a heterogeneous group of pathophysiologic causes, including thrombosis, embolism, and hemorrhage. Strokes currently are classified as either hemorrhagic or ischemic. Acute ischemic stroke refers to strokes caused by thrombosis or embolism and account for 80% of all strokes.
Ischemic strokes result from blockage of the arteries that supply the brain, most commonly in the branches of the internal carotid arteries. The blockage usually results when a piece of a blood clot (thrombus) or of a fatty deposit (atheroma) due to atherosclerosis breaks off (becoming an embolus), travels through the bloodstream, and lodges in an artery that supplies the brain. Blood clots may form when a fatty deposit in the wall of an artery ruptures. The rupture of such a fatty deposit may also form when a large fatty deposit slows blood flow, reducing it to a trickle. Blood that flows slowly is more likely to clot. Thus, the risk of a clot forming in and blocking a narrowed artery is high. Blood clots may also form in other areas, such as in the heart or on a heart valve. Strokes due to such blood clots are most common among people who have recently had heart surgery and people who have a heart valve disorder or an abnormal heart rhythm (arrhythmia), especially atrial fibrillation. Also, in certain disorders such as an excess of red blood cells (polycythemia), the risk of blood clots is increased because the blood is thickened.
An ischemic stroke can also result, if the blood flow to the brain is reduced, as may occur when a person loses a lot of blood or has very low blood pressure. Occasionally, an ischemic stroke occurs when blood flow to the brain is normal but the blood does not contain enough oxygen. Disorders that reduce the oxygen content of blood include severe anemia (a deficiency of red blood cells), suffocation, and carbon monoxide poisoning. Usually, brain damage in such cases is widespread (diffuse), and coma results. An ischemic stroke can occur, if inflammation or infection narrows blood vessels that supply the brain. Similarly, drugs such as cocaine and amphetamines can cause spasm of the arteries, which can lead to a narrowing of the arteries supplying the brain to such an extent that a stroke is caused.
The brain requires glucose and oxygen to maintain neuronal metabolism and function. The inadequate delivery of oxygen to the brain leads to a hypoxia and ischemia results from insufficient cerebral blood flow. The consequences of cerebral ischemia depend on the degree and the duration of reduced cerebral blood flow. Neurons can tolerate ischemia for 30-60 minutes. If flow is not re-established to the ischemic area, a series of metabolic processes ensue. The neurons become depleted of ATP and switch over to anaerobic glycolysis, a much less efficient pathway. Lactate accumulates and the intracellular pH decreases. Without an adequate supply of ATP, ion pumps in the plasma membrane fail. The resulting influx of sodium, water, and calcium into the cell causes rapid swelling of neurons and glial cells. Membrane depolarization also stimulates the massive release of the amino acids glutamate and aspartate, both of which act as excitatory neurotransmitters in the brain. Glutamate further activates sodium and calcium ion channels in the neuronal cell membrane namely the well characterized N-methyl-D-aspartate (NMDA) calcium channel. Excessive calcium influx causes the disordered activation of a wide range of enzyme systems (proteases, lipases, and nucleases). These enzymes and their metabolic products, such as oxygen free radicals, damage cell membranes, genetic material, and structural proteins in the neurons, ultimately leading to the cell death of neurons (Dirnagl, U. et al. (1999) Trends Neurosci, 22: 391-397).
Strokes begin suddenly, develop rapidly, and cause death of brain tissue within minutes to days. In the ischemic brain, we commonly distinguish two tissue volumes—the core of the infarction and the surrounding zone, known as ischemic penumbra—the underperfused and metabolically compromised margin surrounding the irrevocably damaged core. Core and penumbra are characterized by two different kinds of cell death: necrosis and apoptosis (which is also called programmed cell death or delayed neuronal cell death). The severe perfusion deficit in the core causes a breakdown of metabolic processes, cellular energy supply and ion homeostasis, which causes the cells to lose their integrity within minutes. Thus, acute necrosis of cell and tissue prevails in the core. In the penumbra, some residual perfusion is maintained by collateral vessels, which may be unable to maintain the full functional metabolism, but prevents immediate structural disintegration. However, over time (hours to several days), the alteration of cellular homeostasis causes more and more cells to die, and the volume of the infarction increases. The penumbra has thus to be considered as tissue at risk during the materation of the infarct. In this region, apoptosis and inflammatory signaling cascades play an important role. It may initially constitute 50% of the volume that will end up as infarction. The mechanisms that lead to delayed cell death provide targets for a specific neuroprotective therapy in brain regions challenged by ischemia, but which are still viable.
Therapeutic options so far are highly disappointing: Thrombolysis with rtPA, the only therapy with proven efficacy in a major clinical trial (NINDS), is only effective within a three hour time window, limiting its application to only a few percent of patients with ischemic stroke. In other words, besides basic supportive therapy, at present more than 95% of strokes cannot be treated specifically. This is in sharp contrast to our knowledge concerning the basic pathophysiology of this disease, which has emerged over the last decade. In particular, extensive knowledge has accumulated on mechanisms of parenchymal brain damage and endogenous neuroprotection, as well as functional and structural reorganization.
Recently, attention has focused on potential therapeutic roles for endogenous brain proteins possessing neuroprotective properties. EPO, a glycoprotein hormone produced primarily by cells of the peritubular capillary endothelium of the kidney, which is a member of the growth hormone/prolacton cytokine family (Thu Y. and D'Andrea A. D: (1994) Curr. Opin. Hernatal. 1: 113-118) is a promising candidate. Although EPO was first characterized and is now widely known for its role as a haematopoietic hormone the detection of EPO and its receptor (EPOR) in rodent and human brain tissue as well as in cultured neurons and astrocytes expanded the search for other biological roles of EPO.
In the brain, a paracrine EPO/(Epo-R)2 system exists independent of the endocrine system of adult erythropoiesis; neurones express (Epo-R)2 and astrocytes produce EPO (Ruscher et al. (2002) J. Neurosci. 22, 10291-301; Prass et al. (2003) Stroke 34, 1981-1986). It was demonstrated in vitro and in vivo that EPO is a potent inhibitor of neuronal apoptosis induced by ischemia and hypoxia (Ruscher et al. (2002) J. Neurosci, 22, 10291-301; Bemaudin, M., et al. (1999) J Cereb Blood Flow Metab. 19: 643-51; Morishita, E., et al. (1997) Neuroscience. 76: 105-16). It was reported by several groups that addition of EPO to neuronal cultures protects against hypoxic and glutamic acid toxicity (Henn F. A: and Braus D. F. (1999) Eur. Arch. Psychiatry Clin. Neurosci. 249: 48-56, Vogeley K. et al. (2000) Am. J. Psychiatry 157: 34-39) and reduces neurologic dysfunction in rodent models of strike (Brines M. L. et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97: 10526-10531 and Bemaudin et al. (1999) J. Cereb. Blood Flow Metab. 10: 643-.651). The promising results of these experiments have been corroborated in human studies wherein it was shown that EPO therapy for acute stroke is safe and might be beneficial (Ehrenreieh H. et al. (2002) Mol. Medicine. 8: 495-505) and WO 00/35475 A2. These cell and more particular neuroprotective properties of EPO have led to further research in this area to substantiate these findings in larger trial and the use of EPO is now proposed in other indication as well including, for example, schizophrenia (Ehrenreich H et al. (2004) Molecular Psychiatry 9: 42-54 and WO 02/20031 A2).
For the application of EPO to prevent tissue damage the hematopoietic activity is often not required and might be detrimental if large amounts of EPO are administered to treat or atmliorate the effects of, hypoxia or ischemia induced tissue damage. Therefore, attempts have been made to create EPO variants, which only exhibit the cell protective property but not the hematopoietic properties. US 2003/0130197 describes peptide mimetics of EPO for the treatment of neurodegenerative disorders, which bear no sequence homology to naturally occurring EPO or fragments thereof. U.S. Pat. No. 6,531,121 discloses a asialoerythropoietin which is generated by complete desialylation of recombinant EPO showed an increased ability to cross the endothelial cell barrier and had a decreased hematopoietic activity. Carbamylated erythropoietin (CEPO) was also shown to exhibit a tissue protective effect but no erythropoietic effect (Leist et al. (2004) Science 305: 239-242 and WO 2005/025606 A1.
Finally, it was shown that a 17-mer peptide of EPO inhibited cell death of two neuronal cell lines, SK-N-MC and NS20Y (Campana W. M. et al. (1998) Int J. Mol. Medicine 1: 235-241), while at the same time having no hematopoietic activity. However, 1 ng/ml of the EPO peptide was needed to elicit the same antiapoptotic effect as 100 pg/ml recombinant EPO (rhEPO) in NS20Y cells and as 400 pg/ml rhEPO in SK-N-MC cells. Given the apparent molecular weight of rhEPO of about 66.000 g/mol (the calculated molecular weight is about 33.000 g/mol but does not include the weight of oligosaccharide residues comprised in rhEPO) and of about 1.900 g/mol of the EPO peptide a concentration of 1.52 pmol/l and 6.06 pmol/l, respectively, of rhEPO and 1 nmol/l of the EPO peptide elicited the same level of a cell protective effect. Consequently, the EPO peptide is between 650-fold to 165-fold less active than rhEPO in prevention of cell death. It is evident from this figures that the EPO region comprised in the 17-mer does not play a major role in the cell protective function of EPO. Therefore, all EPO variants, which have a decreased hematopoietic activity known in the prior art suffer from the disadvantage that they are not natural occurring since they have either lost their natural glycosylation or they are artificial truncations and/or they have vastly diminished cell protective activity, if compared to rhEPO. Therefore, there is a need in the prior art to provide an EPO derivative, which is close to the naturally occurring EPO and which has the same or better tissue protecting activity as rhEPO but less or no hematopoietic, in particular no erythropoietic activity.
This problem is solved by the provision of new EPO variants, which were found to occur naturally in human and mouse tissue (brain, kidney) and which exhibit a cell protective activity similar or better to rhEPO but which do not exhibit any significant hematopoietic activity.